NMR Study of General Anesthetic Interactions with the Neuronal N-Acetylcholine Receptor Transmembrane Domain

Abstract

General anesthetics have been shown to inhibit neuronal nicotinic acetylcholine receptors (nAChRs) channel function but the mechanisms of this action are not very clear. Here we present the study of the interactions of the anesthetics halothane, isoflurane, and ketamine with the transmembrane domains of the human nAChRs (α4)2(β2)3 and (α4)3(β2)2 using high-resolution solution NMR. The isolated transmembrane domains of α4 and β2 subunits, with the extracellular and intracellular domains removed by mutagenesis, were expressed in E. coli, purified into detergent micelles and mixed in the indicated ratio. Residues affected by anesthetics were determined on the basis of their chemical shift changes in 15N-1H HSQC spectra upon titration by anesthetics. In addition to intrasubunit binding sites identified recently, anesthetic interaction sites at the subunit interfaces were also detected. The main site was located near the extracellular interface involving the beginning of TM1 of one of the subunits and the loop between TM2 and TM3 (or residues near the loop) of the other subunit. Moreover, two leucines in the middle of TM2 (one of which facing the pore is the putative hydrophobic gate of the channel) were also significantly affected by anesthetics. Both interaction sites were common for halothane and isoflurane. The intravenous anesthetic ketamine affected a number of residues in TM4, suggesting that ketamine could bind at the protein-detergent interface. The ketamine binding site at the extracellular interface appeared to be similar to the halothane or isoflurane one. Anesthetics also affected NMR peak intensity and line width of specific residues and in some cases resulted in peak splitting due to conformational exchange, indicating modulations of protein dynamics on the microsecond-to-millisecond timescale, which directly related to protein function. This work was supported by NIH grants: R01GM066358, R01GM056257, and R37GM049202.